Multispecific antigens binding fragments and multispecific antibodies derived therefrom comprising mutant CH1 and CL-κ domains

ABSTRACT

The invention relates to multispecific antibody constructs comprising Fab fragments having mutations at the interface of the CH1 and CL domains, said mutations preventing heavy/light chain mispairing.

The invention relates to the production of multispecific, in particularbispecific, antibody molecules.

Among protein-based drugs, monoclonal antibodies (mAbs) have aparticular characteristic, acting both as a drug and as a targeteddelivery system. Mabs have recently shown a great potential in treatmentof various diseases, including in particular several types of cancer,where they are much more specific than conventional chemotherapy.

The basic structure of a naturally occurring antibody molecule is aY-shaped tetrameric quaternary structure consisting of two identicalheavy chains and two identical light chains, held together bynon-covalent interactions and by inter-chain disulfide bonds.

In mammalian species, there are five types of heavy chains: α, δ, ε, γ,and μ, which determine the class (isotype) of immunoglobulin: IgA, IgD,IgE, IgG, and IgM, respectively. The heavy chain N-terminal variabledomain (VH) is followed by a constant region, containing three domains(numbered CH1, CH2, and CH3 from the N-terminus to the C-terminus) inheavy chains γ, α and δ, while the constant region of heavy chains μ andε is composed of four domains (numbered CH1, CH2, CH3 and CH4 from theN-terminus to the C-terminus). The CH1 and CH2 domains of IgA, IgG, andIgD are separated by a flexible hinge, which varies in length betweenthe different classes and in the case of IgA and IgG, between thedifferent subtypes: IgG1, IgG2, IgG3, and IgG4 have respectively hingesof 15, 12, 62 (or 77), and 12 amino acids, and IgA1 and IgA2 haverespectively hinges of 20 and 7 amino acids.

There are two types of light chains: λ and κ, which can associate withany of the heavy chains isotypes, but are both of the same type in agiven antibody molecule. Both light chains appear to be functionallyidentical. Their N-terminal variable domain (VL) is followed by aconstant region consisting of a single domain termed CL.

The heavy and light chains pair by protein/protein interactions betweenthe CH1 and CL domains, and the two heavy chains associate byprotein/protein interactions between their CH3 domains. The structure ofthe immunoglobulin molecule is generally stabilised by interchainsdisulfide bonds between the CH1 and CL domains and between the hinges.

The clinical efficacy of therapeutic antibodies relies on both theirantigen-binding function and their effector functions, which arerespectively associated with different parts of the immunoglobulinmolecule.

The antigen-binding regions correspond to the arms of the Y-shapedstructure, which consist each of the complete light chain paired withthe VH and CH1 domains of the heavy chain, and are called the Fabfragments (for Fragment antigen binding). Fab fragments were firstgenerated from native immunoglobulin molecules by papain digestion whichcleaves the antibody molecule in the hinge region, on the amino-terminalside of the interchains disulfide bonds, thus releasing two identicalantigen-binding arms. Other proteases such as pepsin, also cleave theantibody molecule in the hinge region, but on the carboxy-terminal sideof the interchains disulfide bonds, releasing fragments consisting oftwo identical Fab fragments and remaining linked through disulfidebonds; reduction of disulfide bonds in the F(ab′)2 fragments generatesFab′ fragments.

The part of the antigen binding region corresponding to the VH and VLdomains is called the Fv fragment (for Fragment variable); it containsthe CDRs (complementarity determining regions), which form theantigen-binding site (also termed paratope). Besides allowing to directspecifically the antibody to its goal, the antigen-binding region mayinduce upon binding to its target antigen a variety of biologicalsignals, which may be positive or negative depending on both thetargeted antigen and the epitope recognised by the antibody on saidantigen. For use in the field of cancer therapy, one generally favoursantibodies delivering a growth-inhibitory or a pro-apoptotic signal,resulting in cytostasis or in death of the tumor cells (VERMA et al., JImmunol, 186, 3265-76; 2011).

The effector function of the antibody results from its binding toeffector molecules such as complement proteins, or to Fc receptors onthe surface of immune cells such as macrophages or natural killer (NK)cells. It results in different effects leading to the phagocytosis orlysis of the targeted antigen, such as antibody dependent phagocytosis(ADP), antibody-dependent cell mediated cytotoxicity (ADCC), orcomplement dependent cell mediated cytotoxicity (CDC).

The effector region of the antibody which is responsible of its bindingto effector molecules or cells, corresponds to the stem of the Y-shapedstructure, and contains the paired CH2 and CH3 domains of the heavychain (or the CH2, CH3 and CH4 domains, depending on the class ofantibody), and is called the Fc (for Fragment crystallisable) region.

The ADCC, ADP, and CDC mediated by the Fc region play a major part inthe therapeutic activity of mAbs. The ADCC mechanism seems to becentral, since it has been demonstrated that in nude mice geneticallydeficient for the Fc gamma receptor, the therapeutic action againsthuman tumour xenografts of the two major clinically successful mAbs,anti-HER2 and anti-CD20, was almost entirely abolished (CLYNES et al.,Nat Med, 6, 443-6, 2000). The ADP mechanism has also been shown to be ofcentral importance in several murine models of human tumors (UCHIDA etal., J. Exp. Med. 199: 1659-69, 2004), and CDC has also beendemonstrated to play a fundamental role in the therapeutic activity ofanti-CD20 in vivo (DI GAETANO et al., J Immunol, 171, 1581-7, 2003).

Due to the identity of the two heavy chains and the two light chains,naturally occurring antibody molecules have two identicalantigen-binding sites and thus bind simultaneously to two identicalepitopes.

In the 1980s, bispecific antibodies having on a same molecule twoantigen-binding sites recognizing two different epitopes and thereforecapable of simultaneous binding to two different targets, were generatedby fusing two cells producing antibodies with distinct specificities(MILSTEIN & CUELLO, Nature, 305, 537-40, 1983). It was shown that suchbi-specific antibodies were able to target effector T cells toward tumorcells (STAERZ et al., Nature, 314, 628-31, 1985).

A wide range of applications for bi-specific antibodies has beendescribed (SONGSIVILAI & LACHMANN, Clin Exp Immunol, 79, 315-21, 1990),including for instance, in the therapeutic field, targeting of effectorcells (cytotoxic T cells, NK cells, and macrophages), or of effectormolecules (toxins, drugs, prodrugs, cytokines, radioisotopes, andcomplement system) and in the diagnostic field, use as reagents inimmunoassays.

Initially, bi-specific antibodies have been prepared by chemicalconjugation, or by use of quadromas resulting from the fusion betweentwo hybridoma cell lines producing two different Mabs. However, chemicalconjugation may occasionally alter the antigen binding sites, resultingin an impairment of the biological properties of the antibody. Thequadroma approach has the drawback that the random pairing of heavy andlight chains from two different antibodies leads theoretically to tenequally possible combinations resulting in a mixture of immunoglobulinmolecules, only one of which is the desired bi-specific product, whichhas to be separated from the mispaired products.

More recently, genetic engineering has become the method of choice toproduce bi-specific antibodies and had led to the development of a widevariety of different recombinant bi-specific antibody formats. Some ofthese bi-specific antibodies are very simple and derive fromsingle-chain Fv (scFv) fragments from two (or more) differentantibodies, associated through an appropriate peptide linker. Theseantibodies are relatively easy to produce, and since they are formed bya single polypeptide chain and contain only the Fv regions of the parentantibodies, there is no problem of mispairing between chains. Howeverthey are smaller than full-length immunoglobulins, and are devoid ofconstant regions, in particular of the Fc region. Although it may beadvantageous in some applications, for instance when one wishes to avoidFc-mediated effects, it is a disadvantage when Fc-mediated effectorfunction such as CDC, ADCC or ADP is desired. Also, due to their smallsize and lack of Fc region, they have a very short half-life in vivo.

Therefore, other bi-specific recombinant antibodies formats, mimickingmore closely the naturally occurring immunoglobulin molecule, and inparticular having a full Fc region, have been designed. They can begrouped into two main formats.

In the first one (IgG scFv), scFv fragments from an antibody A are fusedto the ends (generally the C-terminal ends) of the heavy chains of anantibody B. The resulting antibody having only one type of heavy chain,which contains the VH, CH1, CH2 and CH3 domains of antibody B and the VHand VL domains of antibody A, and one type of light chain which containsthe VL and CL domains of antibody B, mispairing between chains does notoccur. Such a format is described for instance by QU et al. (Blood, 111,2211-9, 2008).

In the second one, the heavy chain and the light chain from an antibodyA are paired with the heavy chain and the light chain from an antibodyB. This format reproduces the bi-specific antibodies produced by thequadromas, and therefore raises similar problems of mispairing. To solvethe problem of mispairing of the heavy chains, it has been proposed tomutate the CH3 domains of the antibodies in order to favour theirheterodimerization (i.e. pairing of heavy chain A with heavy chain B)and to prevent their homodimerization. This was done by the so-called“knob into holes” approach (RIDGWAY et al., Protein Eng, 9, 617-21,1996; U.S. Pat. No. 7,695,936). A “knob” mutation consisting in thereplacement of a small amino-acid by a larger one is introduced at theCH3 dimer interface of the heavy chain of antibody A, resulting in asteric hindrance which prevents homodimerization. Concurrently in orderto promote heterodimerization, a complementary “hole” mutationconsisting in the replacement of a large amino-acid by a smaller one isintroduced into the CH3 domain of antibody B. To solve the problem ofthe heavy chain/light chain mispairing, it has been proposed to useantibodies of different specificities but sharing a common light chain,previously identified from an scFv phage library (MERCHANT et al., NatBiotechnol, 16, 677-81, 1998; U.S. Pat. No. 7,183,076). The drawback ofthis approach is the difficulty in identifying antibodies having acommon light chain.

The inventors have now found that by mutating some key residues at theinterface of the CH1 and CL domains, it is possible to prevent heavychain/light chain mispairing and thus to ensure the desired matching ofthe chains.

They have more specifically found several sets of mutations suitable tothis end. In a first one, a pair of interacting polar interface residuesis exchanged for a pair of neutral and salt bridge forming residues. Thereplacement of Thr192 by a Glu on CH1 chain and exchange of Asn137 to aLys on CL chain was selected. These two mutated residues form a saltbridge, which is presumed to reinforce the specificity of theassociation, whereas an unwanted pairing should be avoided by the lackof sterical and charge complementarity between the wild type and variantchains. Additionally a substitution of Ser114 to Ala on CL chain wasmade to avoid steric clashes with a bigger lysine side chain.

In a second set of mutations the inventors chose to replace the Leu143of the CH1 domain by a Gln residue, while the facing residue of the CLchain, that is Val133, was replaced by a Thr residue. This first doublemutation constitutes the switch from hydrophobic to polar interactions.Simultaneously a mutation of two interacting serines (Ser188 on CH1chain and Ser176 on CL chain) to valine residues was selected to performthe switch from polar to hydrophobic interactions. This exchange of thepolar/hydrophobic character of the interface interactions is expected tokeep the affinity between the mutated CL and CH1 domains unchanged,while decreasing their respective affinity for other wild typecounterparts, thus preventing mispairing by virtue of unfavorableinteractions occurring upon mismatched (variant/wild type) chainscomplexation a pair of interacting apolar residues is exchanged for apair of polar amino acids, while a pair of interacting polar residues issimultaneously exchanged for a pair of hydrophobic residues.

The third and fourth set of mutations are “knob into holes” mutations.More specifically, in the third set of mutations (KH1) the Leu124 andLeu143 of the CH1 domain have been respectively replaced by an Ala and aGlu residue while the Val133 of the CL chain has been replaced by a Trpresidue, and in the fourth set of mutations (KH2), the Val190 of the CH1domain has been replaced by an Ala residue, and the Leu135 and Asn137 ofthe CL chain have respectively been replaced by a Trp and an Alaresidue.

Sequence position numbers used herein for the CH1 and CL domains referto Kabat numbering (Kabat, E. A. et al., Sequences of proteins ofimmunological interest. 5th Edition—US Department of Health and HumanServices, NIH publication n° 91-3242, pp 662,680,689, 1991).

An object of the present invention is therefore a mutated Fab fragmentselected among:

a) a Fab fragment consisting of:

-   -   the VH and VL domains of an antibody of interest;    -   a CH1 domain which is derived from the CH1 domain of an        immunoglobulin by substitution of the threonine residue at        position 192 of said CH1 domain with a glutamic acid residue;        and    -   a CL domain which is derived from the CL domain of an        immunoglobulin by substitution of the asparagine residue at        position 137 of said CL domain with a lysine residue and        substitution of the serine residue at position 114 of said CL        domain with an alanine residue;

b) a Fab fragment consisting of:

-   -   the VH and VL domains of an antibody of interest;    -   a CH1 domain which is derived from the CH1 domain of an        immunoglobulin by substitution of the leucine residue at        position 143 of said CH1 domain with a glutamine residue and        substitution of the serine residue at position 188 of said CH1        domain with a valine residue; and    -   a CL domain which is derived from the CL domain of an        immunoglobulin by substitution of the valine residue at position        133 of said CL domain with a threonine residue and substitution        of the serine residue at position 176 of said CL domain with an        valine residue.

c) a Fab fragment consisting of:

-   -   the VH and VL domains of an antibody of interest;    -   a CH1 domain which is derived from the CH1 domain of an IgG        immunoglobulin by substitution of the leucine residue at        position 124 of said CH1 domain with an alanine residue and        substitution of the leucine residue at position 143 of said CH1        domain with a glutamic acid residue;    -   a CL domain which is derived from the CL domain of an IgG        immunoglobulin by substitution of the valine residue at position        133 of said CL domain with a tryptophane residue;

d) a Fab fragment consisting of:

-   -   the VH and VL domains of an antibody of interest;    -   a CH1 domain which is derived from the CH1 domain of an        immunoglobulin by substitution of the valine residue at position        190 of said CH1 domain with an alanine residue; and    -   a CL domain which is derived from the CL domain of an        immunoglobulin by substitution of the leucine residue at        position 135 of said CL domain with a tryptophane residue, and        substitution of the asparagine residue at position 137 of said        CL domain with an alanine residue.

According to a preferred embodiment, the CH1 domain is derived from aIgG immunoglobulin, advantageously of the IgG1 subtype. The CL domain ispreferably a kappa type. Preferably, for use in human therapy, theimmunoglobulin from which the mutated CH1 and CL domains are derived isa human immunoglobulin.

The VH and VL domains can be derived from any antibody, native orgenetically engineered, recognizing an epitope that one wishes totarget.

The mutated Fab fragments of the invention can be used in anymultispecific antibody construct where it is necessary to prevent heavychain/light chain mispairing.

Advantageously, they are used in a new multispecific antibody constructdesigned by the inventors, comprising one or more multispecificantigens-binding fragment(s) each of which consists essentially oftandemly arranged Fab fragments, separated by appropriate linkers.

An “antigens-binding fragment” is defined herein as a molecule havingtwo or more antigen-binding regions, each recognizing a differentepitope. The different epitopes can be borne by a same antigenicmolecule or by different antigenic molecules.

Therefore, another object of the invention is a multispecificantigens-binding fragment, comprising at least two, and up to five,different Fab fragments selected among:

-   -   a Fab fragment (herein also defined as a: “wild-type Fab        fragment”) comprising wild-type CH1 and CL domains of an        immunoglobulin    -   a mutated Fab fragment (a) as defined above;    -   a mutated Fab fragment (b) as defined above;    -   a mutated Fab fragment (c) as defined above;    -   a mutated Fab fragment (d) as defined above;

each Fab fragment recognizing a different epitope of interest and saidFab fragments being tandemly arranged in any order, the C-terminal endof the CH1 domain of a first Fab fragment being linked to the N-terminalend of the VH domain of the following Fab fragment through a polypeptidelinker. Generally, said polypeptide linker should have a length of atleast 20, preferably at least 25, and still more preferably at least 30,and up to 80, preferably up to 60, and still more preferably up to 40amino-acids.

Advantageously, said polypeptide linker comprises all or part of thesequence of the hinge region of one or more immunoglobulin(s) selectedamong IgA, IgG, and IgD. If the antibody is to be used in human therapy,hinge sequences of human origin will be preferred.

Sequences of the hinge regions of human IgG, IgA and IgD are indicatedbelow:

IgA1 (SEQ ID NO: 1): VPSTPPTPSPSTPPTPSPS IgA2 (SEQ ID NO: 2): VPPPPPIgD (SEQ ID NO: 3): ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTP IgG1 (SEQ ID NO: 4): EPKSCDKTKTCPPCP IgG2 (SEQ ID NO: 5):ERKCCVECPPCP IgG3: (SEQ ID NO: 6) ELKTPLGDTTHTCPRCPfollowed by 0 or 1 to 4 repeats of (SEQ ID NO: 7) EPKSCDTPPPCPRCP. IgG4:(SEQ ID NO: 8) ESKYGPPCPSCP

Said polypeptide linker may comprise all or part of the sequence of thehinge region of only one immunoglobulin. In this case, saidimmunoglobulin may belong to the same isotype and subclass as theimmunoglobulin from which the adjacent CH1 domain is derived, or to adifferent isotype or subclass.

Alternatively, said polypeptide linker may comprise all or part of thesequences of hinge regions of at least two immunoglobulins of differentisotypes or subclasses. In this case, the N-terminal portion of thepolypeptide linker, which directly follows the CH1 domain, preferablyconsists of all or part of the hinge region of an immunoglobulinbelonging to the same isotype and subclass as the immunoglobulin fromwhich said CH1 domain is derived.

Optionally, said polypeptide linker may further comprise a sequence offrom 2 to 15, preferably of from 5 to 10 N-terminal amino-acids of theCH2 domain of an immunoglobulin.

In some cases, sequences from native hinge regions can be used; in othercases point mutations can be brought to these sequences, in particularthe replacement of one or more cysteine residues in native IgG1, IgG2 orIgG3 hinge sequences by alanine or serine, in order to avoid unwantedintra-chain or inter-chains disulfide bonds.

A non-limitative example of a polypeptide linker which can be used in amultispecific antigens-binding fragment of the invention is apolypeptide having the following sequence:EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 9). Said polypeptideconsists of the full length sequence of human IgG1 hinge (SEQ ID NO: 4),followed by the 9 N-terminal amino-acids of human IgG1 CH2 (APELLGGPS,SEQ ID NO: 10), by a portion of the sequence of human IgA1 hinge(TPPTPSPS, SEQ ID NO: 11), and by the dipeptide GG, added to providesupplemental flexibility to the linker.

Optionally, a shorter portion of the N-terminal sequence of the humanIgG1 CH2 domain can be used. Also, a longer portion of human IgA1 hinge,up to its full-length sequence (preferably minus the N-terminal valineresidue) can be used. According to a particular embodiment, said humanIgA1 hinge sequence can be replaced by an artificial sequence,containing an alternation of threonine, serine and proline residues.

For instance, a variant of the polypeptide of SEQ ID NO: 9, which isalso suitable for use in a multispecific antigens-binding fragment ofthe invention is a polypeptide having the following sequence:EPKSCDKTHTCPPCPAPELLPSTPPSPSTPGG (SEQ ID NO: 12). In this polypeptide,the full length sequence of human IgG1 hinge is followed by the 5N-terminal amino-acids of human IgG1 CH2 (APELL, SEQ ID NO: 13), and bythe sequence PSTPPSPSTP (SEQ ID NO: 14).

In case of a multispecific antigens-binding fragment of the invention,comprising more than two different Fab fragments, the polypeptidelinkers separating the Fab fragments can be identical or different.

According to a preferred embodiment of a multispecific antibody of theinvention, it has two identical antigens-binding arms, each consistingof a multispecific antigens-binding fragment as defined above. Theantigens-binding arms can be linked together in diverse ways, dependingon the intended use for the antibody.

If one wishes to obtain an antibody without Fc-mediated effects, theantibody will comprise no Fc region. In this case, the twoantigens-binding arms can be linked together for instance:

-   -   by homodimerization of the antigens-binding arms through the        inter-chain disulfide bonds provided by the polypeptide        linker(s) separating the Fab fragments if said linker contain        cystein residues; and/or    -   through the addition at the C-terminal end of each        antigens-binding arm, of a polypeptide extension containing        cystein residues allowing the formation of inter-chain disulfide        bonds, and homodimerization of said polypeptide extension        resulting in a hinge-like structure; by way on non-limitative        examples, said polypeptide extension may be for instance a hinge        sequence of an IgG1, IgG2 or IgG3;    -   through a semi-rigid linker joining the C-terminal ends of the        heavy chains of the two antigens-binding arms to form a single        polypeptide chain and maintaining said antigens-binding arms at        a sufficient distance between each other

Alternatively, if effector functions such as CDC, ADCC or ADP aredesired, a multispecific antibody of the invention will further comprisea Fc domain providing these effector functions. The choice of the Fcdomain will depend on the type of effector functions which are desired.

In this case, a multispecific antibody of the invention has animmunoglobulin-like structure, comprising:

-   -   two identical multispecific antigens-binding arms as defined        above;    -   the dimerized CH2 and CH3 domains of an immunoglobulin;    -   either the hinge region of an IgA, IgG, or IgD, linking the        C-terminal ends of the CH1 domains of the antigens-binding arms        to the N-terminal ends of the CH2 domains, or alternatively, the        CH4 domains of an IgM or IgE following the CH3 domains, the        C-terminal ends of the CH1 domains of the antigens-binding arms        being in this case linked directly to the N-terminal ends of the        CH2 domains.

Preferably, the CH2 and CH3 domains and either the hinge region or theCH4 domains are derived from a same immunoglobulin or fromimmunoglobulins of the same isotype and subclass as the CH1 domains ofthe antigens binding arm.

The CH2, CH3, and eventually CH4 domains, as well as the hinge regionsfrom native immunoglobulins can be used. It is also possible to mutatethem, if desired, for instance in order to modulate the effectorfunction of the antibody. In some instances, whole or part of the CH2 orthe CH3 domain can be omitted.

The invention also encompasses any protein chain selected among:

-   -   a light chain of a mutated Fab fragment of the invention;    -   a heavy chain of a mutated Fab fragment of the invention;    -   a heavy chain of an antigens-binding fragment of the invention;    -   a heavy chain of an immunoglobulin-like multispecific antibody        of the invention.

Another object of the invention is a polynucleotide comprising asequence encoding a protein chain of the invention. Said polynucleotidemay also comprise additional sequences: in particular it mayadvantageously comprise a sequence encoding a leader sequence or signalpeptide allowing secretion of said protein chain.

The present invention also encompasses recombinant vectors, inparticular expression vectors, comprising a polynucleotide of theinvention, associated with transcription- and translation-controllingelements which are active in the host cell chosen. Vectors which can beused to construct expression vectors in accordance with the inventionare known in themselves, and will be chosen in particular as a functionof the host cell one intends to use.

The present invention also encompasses host-cells transformed with apolynucleotide of the invention. Preferably, said host cell istransformed with a polynucleotide encoding a heavy chain of anantigens-binding fragment or of a multispecific antibody of theinvention, and two polynucleotides encoding two different light chains:a first light chain pairing specifically with a first VH/CH1 region ofsaid heavy chain; a second light chain pairing specifically with asecond VH/CH1 region of said heavy chain and at least one of said lightchains being a light chain of a mutated Fab fragment of claim 1.Optionally said host-cell may additionally be transformed with a thirdpolynucleotide encoding a third light chain different from the first andsecond light chain, and pairing specifically with a third VH/CH1 regionof said heavy chain, and eventually with a fourth polynucleotideencoding a fourth light chain different from the first, second, andthird light chain, and pairing specifically with a fourth VH/CH1 regionof said heavy chain, and possibly with a fifth polynucleotide encoding afifth light chain different from the first, second, third and fourthlight chain, and pairing specifically with a fifth VH/CH1 region of saidheavy chain.

Said polynucleotides can be inserted in a same expression vector, or inseparate expression vectors.

Host cells which can be used in the context of the present invention canbe prokaryotic or eukaryotic cells. Among the eukaryotic cells which canbe used, mention will in particular be made of plant cells, cells fromyeast, such as Saccharomyces, insect cells, such as Drosophila orSpodoptera cells, and mammalian cells such as HeLa, CHO, 3T3, C127, BHK,COS cells, etc.

The construction of expression vectors of the invention and thetransformation of the host cells can be carried out by the conventionaltechniques of molecular biology.

Still another object of the invention is a method for preparing anantigens-binding fragment or an antibody of the invention. Said methodcomprises culturing a host-cell of the invention and recovering saidantigens-binding fragment or antibody from said culture.

If the protein is secreted by the host-cell, it can be recovereddirectly from the culture medium; if not, cell lysis will be carried outbeforehand. The antibody can then be purified from the culture medium orfrom the cell lysate, by conventional procedures, known in themselves tothose skilled in the art, for example by fractionated precipitation, inparticular precipitation with ammonium sulfate, electrophoresis, gelfiltration, affinity chromatography, etc.

The multispecific antibodies of the invention can be used in all theapplications of multispecific antibodies. In particular they can be usedto obtain medicaments useful in a broad range of therapeuticapplications. These medicinal products are also part of the object ofthe invention.

In particular, the multispecific antibodies of the invention can be usedfor the treatment of various diseases by immunotherapy, including forinstance: passive immunotherapy for malignant pathologies,haematological and solid tumors or auto-immune diseases, inflammation,graft rejection, transplantation; active immunotherapy, by modulatinginteraction between different cell populations in particular immunecells during auto-immune diseases or inflammation; adoptiveimmunotherapy combining immune cells with multispecific antibody;internalisation of neutralising antibodies into selected intracellularcompartment.

By way of non-limitative examples:

-   -   multispecific antibodies of the invention directed against        different antigens expressed by a target cells may be used in        order to induce its death by apoptosis, its downregulation or        conversely its activation;    -   multispecific antibodies of the invention directed against        antigens expressed on target and effector cells, may be used in        order to bridge the two types of cells to induce for instance        the killing of the target cells, by the effector cells;    -   multispecific antibodies directed against different soluble        circulating factors for clearing or blocking at the same time        said soluble circulating factors, for instance the simultaneous        clearing of VEGF and PDGF in the course of cancer therapy, or        the simultaneous clearing (or blocking) of different molecules        which inhibits the activity of immunotherapy, such CTLA4,        programme cell death 1 (PD1) or TIM3 or BTLA, or in the field of        anti-inflammatory therapy, the use of multispecific antibody        directed against different inflammatory cytokines, such as Tumor        Necrosis Factor (TNF) and Interleukin 1 beta (IL1-β)

The present invention will be understood more clearly from the furtherdescription which follows, which refers to non limiting examples of thepreparation and properties of a recombinant bi-specific antibody inaccordance with the invention.

EXAMPLE 1 Design of an Anti-CD5/Anti-HLA-DR Bi-Specific Antibody

Design of Mutant Fab Fragments

The antibodies chosen for the construction of bi-specific antibodies arean anti-CD5 antibody and an anti-HLA-DR antibody both described in PCTWO 2010/145895.

Under their native form, these antibodies were murine monoclonalantibodies (mAbs), of the IgG2a and IgG1 isotypes, respectively, withkappa light chains. Both mAbs were previously transformed into chimericmouse/human mAbs with the constant domains of the heavy chain being ofhuman IgG1 subclass and the constant part of the light chains being ofkappa type, while the variable domains of both chains remained of mouseorigin.

Two different sets of complementary mutations were brought at chosensites of CH1 and CL chains of the anti-CD5 antibody, the anti-HLA-DRantibody remaining under its native form.

The mutation sites in the anti-CD5 antibody were chosen to be importantfor CL/CH1 binding, while preserving the most essential residuesinvolved in the proper folding.

The following approaches called “Charged residues” and“Hydrophobicity-polarity-swap” were used.

In the “Charged residues” approach, a pair of interacting polarinterface residues was exchanged for a pair of neutral and salt bridgeforming residues. The introduction of a salt bridge was hypothesised toreinforce the specificity of the association, whereas an unwantedpairing should be avoided by the lack of sterical and chargecomplementarity between the wild type and variant chains. Afterextensive in silico testing the replacement of Thr192 by a Glu on CH1chain and exchange of Asn137 to a Lys on CL chain were selected. Thesetwo mutated residues thin). a salt bridge. Additionally a substitutionof Ser114 to Ala on CL chain was made to avoid steric clashes with abigger lysine side chain. The resulting mutant is designated hereinafteras the “CR3 mutant”.

For the “Hydrophobicity-polarity-swap” approach, the modified constantdomains were obtained by introducing a quadruple mutation (doublemutation on each chain). This modification swaps the nature of tworesidue-residue interactions on the IgG CH1/CL interface. A pair ofinteracting apolar residues is exchanged for a pair of polar aminoacids, while a pair of interacting polar residues is simultaneouslyexchanged for a pair of hydrophobic residues. This exchange of thepolar/hydrophobic character of the interface interactions washypothesised to keep the affinity between the mutated CL and CH1 domainsunchanged, while decreasing their respective affinity for other wildtype counterparts, thus preventing mispairing by virtue of unfavorableinteractions occurring upon mismatched (variant/wild type) chainscomplexation

After in silico testing of many potential mutations we chose to replacethe Leu143 of the CH1 domain by a Gln residue, while the facing residueof the CL chain, i.e. Val133, was replaced by a Thr residue. This firstdouble mutation constitutes the switch from hydrophobic to polarinteractions. Simultaneously a mutation of two interacting serines(Ser188 on CH1 chain and Ser176 on CL chain) to valine residue wasselected to perform the switch from polar to hydrophobic interactions.The resulting mutant is designated hereinafter as the “mut4 mutant”.

The selected mutations are summarized on Table I below:

TABLE I Modifications CH1 of Heavy chain CL of Light chain Chargedresidues Thr192Glu Asn137Lys and CR3 mutant Ser114AlaHydrophobicity-polarity swap Leu143Gln and Val133Thr and mut4 mutantSer188Val Ser176Val

Other mutations were performed using the “knob into holes” approach(RIDGWAY et al., Protein Eng, 9, 617-21, 1996).

These mutations are summarized on Table II below:

TABLE II CH1/Heavy chain CL/Light chain KH1 Leu124Ala and Leu143GluVal133Trp KH2 Val190Ala Leu135Trp and Asn137Ala

The mutated complexes binding free energies were evaluated using theMM-GBSA method. At the same time, the mispaired complexes models werecreated and their interaction energies were calculated using the samemethodology. For the chosen modifications, the complex between themodified CL and CH1 chains was estimated to be as stable as the wildtype complex, whereas significantly unfavorable interactions in themispaired complexes were observed.

Design of a Polypeptide Linker

A polypeptide linker was designed to link the C-terminus of the CH1region of the anti-HLADR antibody to the N-terminus of the VH region ofthe mutant anti-CD5 antibody.

This polypeptide linker comprises a full-length IgG1 hinge region,followed by the 9 N-terminal amino-acids of human IgG1 CH2, by a portionof the sequence of human IgA1 hinge, and by the dipeptide GG. It has thefollowing sequence:

EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 9)

EXAMPLE 2 Construction of a Recombinant Baculovirus ExpressingAnti-HLADR (mAb1) and Anti-CD5 (mAb2) Bi-Specific Antibody

The bi-specific antibody has been expressed and produced using thebaculovirus/insect cells expression system.

This production required the synthesis of a modified heavy chaincomprising the VH/CH1/Hinge domain from a mAb 1 fused to the full lengthheavy chain of a mAb2 and separated by a linker comprising for example(in our current construct) the lower hinge extended with a peptidederived form the natural hinge of human IgA1+GG.

The two different light chains, one specific of the first antibody andthe other one specific of the second antibody are synthesizedindependently and will be paired to the relevant heavy chains, thanks tothe reciprocal mutations introduced into the different CL and CH1 domainas described above.

It could be easier to construct two different baculoviruses the firstone expressing the fused-heavy chain and only one light chain and thesecond one expressing the fused-heavy chain and the second light chainand to co-infect insect cells with the mixture. However, this approachis longer and it would be very difficult to control the stoichiometry ofeach partner. So we decided to construct only one recombinant virusexpressing the fused-heavy chain and the two light chains.

This required the introduction in said baculovirus of two identicalsequences for the CL domains, and two identical sequences for theCH1-hinge (CH1+Hg) domains of the heavy chain. This identity may inducea homologous recombination leading to a reorganisation of the genome andto the loss of genetic information.

In order to avoid this phenomenon, we have introduced only one wild typecoding sequence, the second one being synthetic (modification of allcodons). In this latter, the DNA sequence is different from the originalone but encodes a protein identical (100%) to the wild type one.

2.1 Construction of the cDNA Encoding the Fused-heavy Chain

Anti-HLADR Fab+Linker

A synthetic gene encoding the CH1 domain of the anti-HLADR antibodyfused to the polypeptide linker of SEQ ID NO: 9 was constructed usinghybridization of synthetic overlapping oligonucleotides.

After cloning in a pUC plasmid and control of the sequences, thissynthetic gene was introduced, instead of the wild type sequence in theplasmid pOCγ1KCH1SII/LinkerA1PstI/VHanti-HLADR, between the sequenceencoding the anti-HLADR VH domain and the sequence encoding theextension peptide described in Example 1 above. The resulting plasmid isnamed pOCγ1KCH1εlinkerA1/VH.

Mutated Anti-CD5 Fab:

In order to ensure the proper pairing between heavy and light chains,mutations CR3, mut4, (KH1 or KH2) were introduced in the CH1 domain ofthe anti-CD5 Fab moiety. Plasmid pUCCγ1mutT192E (i.e. for CR3 mutant)was digested with NheI/BstXI and the fragment bearing the mutatedsequence was purified and inserted in pUCKPSCγ1/VHCD5 digested withNheI/BstXI giving pUCKPSCγ1/VHCD5-CR3.

In the same way, pUCKPSCγ1/VHCD5-mut4, (pUCKPSCγ1/VHCD5-KH1 andpUCKPSCγ1/VHCD5-KH2) were constructed.

Full-length Fused-heavy Chain:

The cDNA encoding the full-length fused-heavy chains were constructedgiving pVTanti-HLADR/linkerA1/antiCD5/CR3,pVTanti-HLADR/linkerA1/antiCD5/mut4,(pVTanti-HLADR/linkerA1/antiCD5/KH1, pVTanti-HLADR/linkerA1/antiCD5/KH2)respectively.

The resulting transfer vectors are namedpVTanti-HLADR/linkerA1/antiCD5/CR3 andpVTanti-HLADR/linkerA1/antiCD5/mut4 (pVTanti-HLADR/linkerA1/antiCD5/KH1,and pVTanti-HLADR/linkerA1/antiCD5/KH2) respectively.

2.2 Construction of the cDNA Encoding the Light Chain

Construction of a New Transfer Vector

The production of a fully functional bi-specific antibody requires thecoexpression of (i) a fused-heavy chain as described above, and of (ii)two light chains, the light chain specific of anti-CD5 bearing themutations (CR3, mut4, KH1 or KH2) and the light chain of the anti-HLADR(Mab1). This necessitates choosing a third locus, besides the classicpolyedrin and p10 loci, to insert a third coding sequence chain into thebaculovirus genome. A locus called “gp37” (CHENG et al. J. Gen. Virol.,82, 299-305, 2001) which is not essential for baculovirus replicationtherefore allowing the insertion of a foreign gene, was chosen.

A new transfer vector (pVTgp37) containing a unique XbaI cloning siteunder control of a synthetic P10 promoter, flanked by gp37 sequences wasconstructed.

Construction of a Synthetic CL Domain

As described for the reconstitution of the CH1 domain of the heavychain, the synthetic CL domain was synthesized using overlappingsynthetic oligonucleotides. Two sub-fragments were generated, CKFr1 andCKFr2.

After cloning in a pUC plasmid and control of the sequences, CKFr1 andCKFr2 were introduced, instead of the wild type sequence encoding the Cκdomain, in the plasmid pUCK/VLanti-HLADR

Introduction of the Light Chain in the Gp37 Transfer Vector

The reconstituted sequence encoding the light chain containing thesynthetic constant domain Cκ was isolated after digestion with XbaI andintroduced in transfer vector pVTgp37 at the unique XbaI site, givingthe final construct pVTgp37P10S1CKεVLanti-HLADR.

2.3 Construction of Recombinant Viruses

Construction of a recombinant virus expressing the bi-specific antibodyrequires two steps: (i) the construction of a first baculovirusexpressing only the light chain of Mab 1, the anti-HLADR (ii) theconstruction of the virus expressing the bi-specific antibody,anti-CD5/anti-HLADR.

Construction of a Recombinant Virus Expressing the Anti-HLADR

For this purpose, Sf9 cells were cotransfected withpVTgp37P10S1CKεVL/anti-HLADR and with DNA extracted from a modifiedbaculovirus expressing the polyhedrin gene under the control of the gp37promoter at the gp37 locus.

Recombinant viruses exhibiting a “polyhedrin negative” phenotype wereisolated and the genome of four recombinant viruses was controlled bySouthern blot using the synthetic kappa c-DNA as a probe. Onerecombinant virus called BacLC/anti-HLADR was selected.

Construction of Recombinant Viruses Expressing the Bi-specific Antibody

Sf9 cells were cotransfected with transfer vectors bearing the cDNAencoding fused-heavy chains pVTanti-HLADR/linkerA1/anti-CD5 (CR3, mut4,KH1 or KH2) and transfer vectors bearing the cDNA encoding the Mab2light chains pVTVLIICD5CkmutCR3, pVTVLIICD5Ckmut4, (pVTVLIICD5CkKH1 orpVTVLIICD5CkKH2) in the presence of viral DNA extracted fromBacLC/anti-HLADR. Productive clones were screened by ELISA. The genomeof recombinant viruses was controlled by Southern blot using cDNAsencoding human constant γ1 and constant κ region respectively as probes.Two of the selected clones (clone C683 for anti-CD5/anti-HLADR(CR3) andclone C977 for anti-CD5/anti-HLADR(mut4) were used for the production ofantibodies.

2.4 Production and Purification of the Recombinant Antibodies

Sf9 cells were seeded at a density of 600,000 cells/ml in 400 ml ofserum free medium in roller bottles and infected with either clone C683or clone C977 at a multiplicity of infection of 2 PFU per cell. After 4days incubation at 28° C., the supernatant was collected and secretedrecombinant antibodies were purified on protein A SEPHAROSEchromatography resin (GE, HealthCare). The concentration of purifiedbi-specific antibodies was determined by using BCA assay, as recommendedby the manufacturer PIERCE, and with bovine IgG (ref Standard PIERCE23209) as a standard.

The structure of the final bi-specific antibodies is shown on FIG. 1.

Legend of FIG. 1: Mab 1: anti-HLA-DR Fab; Mab2: anti-CD5 mutant Fab;Linker: polypeptide linker; Hinge: human IgG1 hinge; Fc human IgG1 Fcregion. Due to the presence of 2 cysteine residue(s) from IgG1 hinge thetwo antigens-binding arms are connected through two interchaindisulfide(s) bridge(s).

The molecular weight of the purified anti-CD5/anti-HLADR mutantantibodies was evaluated on SUPEROSE 6chromatography colums (GEHealthCare). More than 90% of the molecules purified on Protein ASEPHAROSE presented an estimated molecular weight of about 299 kDa onSUPEROSE 6, thus correlating with the theoretical molecular weight of260 kDa (MW calculated without glycans) for the recombinant bi-specificantibody of FIG. 1.

These antibodies were further analysed by SDS-PAGE under reducing ornon-reducing conditions. The results are shown on FIG. 2.

Legend of FIG. 2: (A) Samples analyzed in reducing conditions; (B)Samples analyzed in non-reducing conditions; BS: bi-specific antibody;Mab: control IgG1 recombinant anti-HLADR.

The size of the heavy chain of the bi-specific antibodies estimated onthis gel corresponds to the calculated molecular weight of 78 000 Da ofthe fused-heavy chain of the antibody of FIG. 1.

These analyses indicate that the methodology described here leads to theformation of a molecule resulting of the association of two fused-heavychains with two couples of light chains

EXAMPLE 3 Functional Properties of Anti-CD5/Anti-HLADR (CR3)

Functionality of the Binding Sites

It was important to show that the bi-specific antibodies were able tobind through their 2 different antibody binding sites. To that aim wetested their binding to cells that expressed either CD5 or HLADR by flowcytometry. Briefly all antibodies under study were coupled withphycoerythrin (PE), using the Zenon R-phycoerythrin human (or mouse) IgGlabelling kit. Cell lines were then incubated with PE-labelled anti-CD5,anti-HLADR, CR3 bispecific antibodies or control mouse or human IgG1irrelevant antibodies, washed and then analysed by flow cytometry.

The results of the binding to the CD5⁺/HLADR⁻ Jurkat cell line and tothe CD5⁻/HLA-DR⁺JOK1 cell line are shown on FIGS. 3 and 4, respectively.

Legend of FIG. 3: The Jurkat cell line (CD5+/HLADR−) was stained withPE-labelled mouse anti-CD5 (anti-CD5m), mouse anti-HLADR (anti-HLADRm),bispecific anti-CD5/anti-HLADR CR3 chimeric antibody or control human ormouse IgG1 antibodies (hIgG1 and mIgG1, respectively), all PE-labelled.Cells were then analysed by standard flow cytometry. The overlayedhistograms for each antibody are shown, with mean fluorescence intensityvalues (MFI) indicated in parentheses for each antibody. 1. mIgG-PE 1 μg(MFI=2.6); 2. Anti-CD5m-PE 1 μg (MFI=17); 3. Anti-HLADRm-PE 1 μg(MFI=2.5); 4. hIgG1-PE 1 μg (MFI=3.9); 5. Anti-CD5/anti-HLADR/chi-PE CR31 μg (MFI=103).

Legend of FIG. 4: The JOK1 cell line (CD5−/HLADR+) was stained withPE-labelled mouse anti-CD5 (anti-CD5m), mouse anti-HLADR (anti-HLADRm),bispecific anti-CD5/anti-HLADR CR3 chimeric antibody or control human ormouse IgG1 antibodies (mIgG1 and hIgG1, respectively), all PE-labelled.Cells were then analysed by standard flow cytometry. The overlayedhistograms for each antibody are shown, with mean fluorescence intensityvalues (MFI) indicated in parentheses for each antibody. 1. mIgG1-PE 1μg (MFI=25); 2. (hatched) Anti-CD5m-PE 1 μg (MFI=18); 3. Anti-HLADRm-PE1 μg (MFI=432); 4. hIgG1-PE 1 μg (MFI=3); 5. Anti-CD5/anti-HLADR/chi-PECR3 1 μg (MFI=3521).

FIG. 3 shows that both mouse anti-CD5 and bi-specific CR3 are able tobind to the CD5⁺ Jurkat cell line, whereas anti-HLADR antibody does not,as expected. Thus bi-specific CR3 antibody recognises the CD5 antigen onCD5 positive cell line.

FIG. 4 shows that mouse anti-HLADR and bi-specific CR3 antibodies bindwith high intensity to the CD5⁻/HLADR⁺ JOK cell line, whereas mouseanti-CD5 does not, as expected. This demonstrates that the bi-specificCR3 antibody recognises the HLADR antigen on a HLADR+ cell line.

We conclude that the bi-specific CR3 antibody recognises bothspecificities (CD5 and HLADR) correctly.

Binding to Antigens Expressed on a Same Cell

Next we wanted to document further that bi-specific CR3 antibody whichwe showed was able to bind to its 2 targets when they were expressed onthe same cell surface, that is in cis. To this aim we first identified aB-CLL patient sample which expressed approximately the same amounts ofCD5 and HLADR. B-CLL patients cells were incubated with mouse anti-CD5,mouse anti-HLADR or mouse IgG1 control antibody for 30 minutes a roomtemperature and then with FITC-labelled anti-mouse IgG secondaryantibody. After washing, cells were analysed by standard flow cytometry.As shown in FIG. 5A, the cells expressed similar amounts of CD5 andHLADR, with mean fluorescence intensities of 65 and 98, respectively.

In order to demonstrate that bi-specific anti-CD5/anti-HLADR CR3antibody bound both antigens on the same cells, we then performed across-blocking experiment on the same B-CLL sample. Cells were incubatedwith 1 μg/ml chimeric CR3 bi-specific antibody, in presence or absenceof excess (10 μg/ml) mouse anti-CD5 or mouse anti-HLADR antibodies orboth. After washing, binding of bi-specific CR3 antibody was detected byincubation with a secondary monoclonal FITC-labelled antibody(Sigma-Aldrich), specific for human Fc, and unable to bind to mouse Fc(data not shown).

Legend of FIG. 5:

Panel A: B-CLL patient cells were incubated with mouse anti-CD5 (mCD5),mouse anti-HLADR (mDR) or mouse irrelevant IgG antibody (mIgG) ascontrol. After washing, cells were stained with FITC-labelled anti-mousesecondary antibody and then analysed by standard flow cytometry. The MFIfor mCD5 and mDR are indicated between brackets.

Panel B: Cells from the same patient as in A were incubated with 1 μg/mlchimeric CR3 alone (dark thick line) or in presence of 10 μg/ml mouseanti-CD5 (light grey line) or mouse anti-HLADR (dark grey line) or both(discontinuous line). After washing, cells were incubated withmonoclonal FITC-labelled anti human Fc antibody, washed and analysed byflow cytometry. The overlayed histograms for each condition are shownwith MFI obtained in each case indicated above each curve. BS:Bi-specific, m: mouse, h: human, chi: chimeric.

MFI  

  Negative control (anti-human IgG-FITC) 6  

  chBI-CR3 1 ug/ml 97  

  chBI-CR3 1 ug/ml + mCD5 10 ug/ml 72  

  chBI-CR3 1 ug/ml + mDR 10 ug/ml 54  

  chBI-CR3 1 ug/ml + mCD5 + mDR 10 ug/ml 20

As shown in FIG. 5B, bi-specific CR3 antibody alone resulted in a meanfluorescence intensity (MFI) of 97. Competition by anti-CD5 oranti-HLADR alone only partially displaced CR3 (MFI 72 and 54,respectively). In contrast, adding both antibodies together displacedbi-specific CR3 antibody nearly completely (MFI 20). These data suggestthat bi-specific CR3 antibody binds to the cells via either the CD5 orHLADR moiety and displacing it requires competition by a mixture ofanti-CD5 and anti-HLADR antibodies.

We conclude from these data that the bi-specific antibody CR3, thechimeric anti-CD5/anti-HLADR antibody can bind to both HLADR and CD5 onthe same cell.

EXAMPLE 4 Functionality of the Fc Moiety and of the Antigens-BindingMoiety of the Bi-Specific Antibodies

Fc Moiety

The Fc moiety of antibody molecules are capable of activating variousimmune functions such as phagocytosis (ADP) and antibody dependentcytotoxicity (ADCC) by binding to FcγRs on macrophages (FcγRI, II andIII) and NK cells (FcγRIII), respectively. Since the constructedbi-specific antibodies have an Fc moiety derived from human IgG1, wehave tested whether it is functional and therefore able to mediate theseimmune mediated functions.

Antibody-Dependent Cellular Cytotoxicity (ADCC) by Natural Killer Cells

First we wanted to determine whether the Fc part of the bi-specific CR3molecule was active when either of its paratopes was binding to itsrespective molecule. We analyzed the ADCC, mediated by Fc binding to NKcells, induced on a CD5⁺/HLA-DR⁻ target such as Jurkat cells and onHLA-DR⁺/CD5⁻ targets such as JOK1 and double positive targets JOK1.5.3cells. NK cells were purified from peripheral blood mononuclear cells byimmunobead selection. Target cells were labelled with 1 μMcarboxyfluorescein diacetate succinimidyl ester (CFSE) at 4° C. for 20minutes, washed and cultured with purified NK cells at 37° C. for 4hours at a 10:1 effector to target ratio (E:T). Cells were then labelledwith 7AAD and analysed by flow cytometry. Percentage killing wasmeasured as percent 7AAD positive targets (CFSE+) with respect to totalCFSE+ cells.

The results are shown in FIG. 6.

Legend of FIG. 6: JURKAT (CD5⁺HLADR⁻, panel A), JOK1 (CD5⁻HLADR⁺, panelB) and JOK1 5.3 (CD5⁺HLADR⁺, panel C) were CFSE labelled and used inADCC assays in presence of human NK cells at 10:1 E:T ratio and 1 μg/mlchimeric anti-CD5 (anti-CD5chi) or chimeric anti-HLADR (anti-HLADRchi)or 2 μg/ml bi-specific CR3 antibody. Cytotoxicity was measured by flowcytometry after 4 hours at 37° C.

The data show that the bi-specific CR3 antibody mediates ADCC on all 3cell lines (33-78% cytotoxicity). In contrast anti-CD5chi andanti-HLADRchi are cytotoxic only for CD5⁺ or HLADR⁺ cell lines,respectively.

We conclude that the Fc moiety of bi-specific CR3 antibody is functionalallowing the antibody to mediate ADCC of targets expressing either CD5,HLADR or both antigens.

Phagocytosis

In order to confirm the functionality of Fc part of CR3 molecule withrespect to binding to FcγRs present on macrophages (FcγRI, FcγRII andFcγRIII), and simultaneous binding of its paratopes to their respectivemolecules, we assessed ADP in vitro. CD 14⁺ monocytes were purified fromhealthy donors' mononuclear cells by anti-CD14 microbeads magnetic cellsorting, according to the manufacturer's instructions (Miltenyi Biotec).They were cultured in 8-well chamber slides (LabTek; Nunc) at 2×10⁵/wellfor 6-7 days in RPMI 1640 medium supplemented with 20% foetal bovineserum and 20 ng/ml human rM-CSF (R&D Systems). Phagocytosis of B-CLLtarget cells (CD5⁺/HLA-DR⁺) by these macrophages was then performed. Atotal of 2×10⁵ B-CLL targets was added in each well in presence orabsence of 0.01 to 0.1 μg/ml CR3 bi-specific antibody or anti-CD20 mAbrituximab. After 2 h at 37° C., slides were gently rinsed in PBS, fixed,and stained with May-Gruenwald Giemsa. Phagocytosis was evaluated bycounting under the microscope at least 200 cells for each experimentalcondition, using the ImageJ 1.38 image processing and analysis software,and calculating the percentage of macrophages that engulfed at least onetumor target cell with respect to total macrophages.

The results are shown on FIG. 7.

Legend of FIG. 7: Percentage phagocytosis is shown in the Y-axis and theconcentrations of antibody used are shown in the X-axis, ranging from0.01 to 1 μg/ml of bi-specific antibody CR3 or monospecific anti-CD20antibody rituximab (RTX). 0: no antibody added.

The data below show that the bi-specific CR3 antibody mediates about 40%phagocytosis above background at 0.1-1 μg/ml, similarly to Rituximab.Negative control antibody trastuzumab (anti-HER2) does not mediatephagocytosis (data not shown).

Thus we conclude that the Fc moiety of bi-specific CR3 antibody moleculeis functional and can mediate phagocytosis of target cells bymacrophages through interaction of Fc with FcγRs on these cells.

Antigens-binding Moiety

Redirected by Cytokine Induced Killer Cells by Bi-Specific CR3 Antibody

We then determined whether the paratopes of bi-specific CR3 antibody canbind to its target antigens present on 2 different cell types.

Cytokine induced killer cells (CIK) are activated CD3⁺CD56⁺ doublepositive T lymphocytes generated in vitro by stimulation of peripheralblood mononuclear cells with interferon-gamma, anti-CD3 and expansion invitro for 3-4 weeks with interleukin-2 (SCHMIDT WOLFF et al. J. Exp.Med. 174:139-149; 1991) CIK cells have significant natural cytotoxicactivity against tumor but not normal cells in vitro, similarly to NKcells. CIK cells however do not express FcγR and therefore do notmediate ADCC in presence of mono-specific IgG antibodies such asrituximab. CIK cells express CD5. For this reason CIK cells can beredirected towards HLADR positive but not negative tumor cells bybi-specific antibody CR3 which recognizes CD5 on CIK and HLA-DR on tumortarget. Differently from ADCC, this redirected killing uses the two Fabspecificities of the antibody and not the Fc portion.

Method

Peripheral blood mononuclear cells were cultured at 3×10⁶/ml inserum-free hematopoietic cell medium X-VIVO 15 medium (BioWhittaker,Walkersville, Md., USA) with 1000 U/mL IFN-γ (Gammakine; BoehringerIngelheim, Vienna, Austria) added on day 0.50 ng/mL anti-CD3 (OKT-3,Janssen-Cilag S.p.a., Italy) added on day 1 and 500 U/mL rhIL-2 includedin the medium from day 1 onwards. Expansion was performed for 21-28 daysadjusting cells to 1×10⁶/ml in fresh rhIL-2 containing medium every 3-4days. At the end of the expansion, CD3⁺/CD5⁺/CD56⁺ cytotoxic CIK cellswere 40-70% of the population. Remaining cells are mostly CD3⁺/CD56⁻ CIKprecursor cells.

The human tumor target cell lines BJAB (CD5⁻/HLA-DR⁺), JOK1.5.3(CD5⁺/HLA-DR⁺), Jurkat (CD5⁺/HLA-DR⁻) and KCL22 (CD5⁻/HLA-DR⁻) weremaintained in RPMI-1640 medium (Lonza, Basel, Switzerland) supplementedwith 10% foetal bovine serum (Euroclone, Wetherby, West Yorkshire,U.K.), 2 mM L-glutamine (Euroclone) and 110 μM gentamycin (PHT Pharma,Milano, Italy).

For redirected cytotoxicity assays, target cell lines were labelled for30 minutes at 37° C. with 3.5 μM Calcein-AM (Fluka, Sigma-AldrichCompany, Ayrshire, UK). After washing labelled target cells weredistributed in 96-well plates at 5×10³/well. CIK cell were added at a10:1 effector to target ratios in presence or absence of 1 μg/mlbi-specific CR3 antibody. After 4 hours, the cells were sedimented bycentrifugation, 100 μl supernatant were collected and calcein releasewas determined using a fluorescence microplate reader (GENios, TECAN,Austria GmbH, Salzburg, Austria) with excitation at 485 nm and emissionat 535 nm. The percentage (%) specific lysis was calculated as: (testcalcein release−spontaneous calcein release)×100/(maximal calceinrelease−spontaneous calcein release). Maximal lysis was achieved byadding 1% Triton X-100 (a nonionic surfactant).

The results are shown on FIG. 8.

Legend of FIG. 8: Calcein-AM loaded target cell lines BJAB, JOK1.5.3,Jurkat and KCL22 were incubated in presence (open bars) or absence(black bars) of 1 μg/ml bi-specific CR3 antibody and in presence of CIKcells at a 10:1 effector:target ratio. After 4 hours, supernatants werecollected and released calcein measured. The data show the measuredpercentage lysis (Y-axis) as means and standard deviations of 2-6separate experiments with each cell line. CTRL: Control withoutantibody.

The results show that in vitro, percentage killing (lysis) of HLADRpositive (BJAB, JOK15.3), but not HLADR negative targets (Jurkat,KCL22), is increased by 50-60% by addition of 1 μg/ml CR3 antibody inpresence of CIK cells at a 10:1 effector:target ratio. This demonstratesthat the intercellular bridge formed by the bi-specific CR3 antibodydramatically enhances killing of the HLADR⁺ targets by CIK cells. Noenhancement of killing of HLADR negative targets is observed,demonstrating specificity.

The specificity of the enhancement by the bi-specific CR3 antibody ofthe cytotoxic effect of CIK cells with respect to normal T cells wasfurther demonstrated as follows: HLADR⁺ BJAB target cells were incubatedwith different amounts of peripheral blood mononuclear cells as effectorcells at effector:target ratios ranging form 1:1 to 10:1 in presence orabsence of 1 μg/ml CR3. Lysis was measured at 4 hours.

The results are shown on FIG. 9.

Legend of FIG. 9: Cytotoxicity experiments were performed with PBMC aseffectors and BJAB as target cells at different effector:target ratios,in presence (black circles) and absence (open circles) of bi-specificantibody CR3. X-axis: Effector:target ratio; Y-axis percent of lysis;CR3: bi-specific CR3 antibody; CTRL: control without antibody.

No effect of the CR3 antibody on lysis of the HLADR⁺ targets by normal Tis observed.

These results show that the divalent bi-specific antibody CR3 can beused in conjunction with cytokine induced killer (CIK) cells in adoptiveimmunotherapy treatment. In this case the different specificities of the2 Fab pairs are used, one pair (in this case anti-HLADR) recognizingtarget cell and the other (anti-CD5) the effector CIK cells. Theseresults indicate that different target antigens could be inserted suchas HER1, HER2, EpCAM, CD19, CD20 or others, in place of HLADR. Theresults also indicate that other antigens expressed by effector cellscould be used in place of CD5, such as CD3 expressed by T lymphocytes,FcγRIII or NKG2D present on NK cells or FcγRI-III on macrophages in theframe work of different forms of cancer therapy.

Redirected Killing by Cytokine Induced Killer Cells by Bi-Specific MUT 4Antibody

We determined whether the bi-specific MUT 4 antibody can bind to itstarget antigens present on 2 different cell types and redirect thekilling of CD5+cytokine induced killer cells (CIK) towards a HLADR+lymphoma target (JOK1 5.3). CIK are activated CD3⁺CD56⁺ double positiveT lymphocytes generated in vitro by stimulation of peripheral bloodmononuclear cells with interferon-gamma, anti-CD3 and expansion in vitrofor 3-4 weeks with interleukin-2 (SCHMIDT WOLFF et al. J. Exp. Med.174:139-149; 1991) CIK cells have significant natural cytotoxic activityagainst tumor but not normal cells in vitro, similarly to NK cells. CIKcells however do not express FcγR and therefore do not mediate ADCC inpresence of mono-specific IgG antibodies such as rituximab. CIK cellsexpress CD5. For this reason CIK cells can be redirected towards HLADRpositive but not negative tumor cells by bi-specific antibody MUT 4which recognizes CD5 on CIK and HLADR on tumor target. Differently fromADCC, this redirected killing uses the two Fab specificities of theantibody and not the Fc portion.

Method

Peripheral blood mononuclear cells were cultured at 3×10⁶/ml inserum-free X-VIVO 15 medium (a cell medium from BioWhittaker,Walkersville, Md., USA) with 1000 U/mL IFN-γ (Gammakine; BoehringerIngelheim, Vienna, Austria) added on day 0.50 ng/mL anti-CD3 (OKT-3,Janssen-Cilag S.p.a., Italy) added on day 1 and 500 U/mL rhIL-2 includedin the medium from day 1 onwards. Expansion was performed for 21-28 daysadjusting cells to 1×10⁶/ml in fresh rhIL-2 containing medium every 3-4days and. At the end of the expansion, CD3⁺/CD5⁺/CD56⁺ cytotoxic CIKcells were about 50% of the population. Remaining cells are mostlyCD3⁺/CD56⁻ CIK precursor cells.

The human tumor target cell line JOK1.5.3 (CD5⁺/HLADR⁺) was maintainedin RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10%foetal bovine serum (Euroclone, Wetherby, West Yorkshire, U.K.), 2 mML-glutamine (Euroclone) and 110 μM gentamycin (PHT Pharma, Milano,Italy).

For redirected cytotoxicity assays, target cell lines were labelled for30 minutes at 37° C. with 3.5 μM Calcein-AM (Fluka, Sigma-AldrichCompany, Ayrshire, UK). After washing labelled target cells weredistributed in 96-well plates at 5×10³/well. CIK cell were added at a10:1 effector to target ratios in presence or absence of 1 or 5 μg/mlbi-specific MUT 4 antibody, CR3 antibody or rituximab (RTX) as controls.After 4 hours, the cells were sedimented by centrifugation, 100 μlsupernatant were collected and calcein release was determined using afluorescence microplate reader (GENios, TECAN, Austria GmbH, Salzburg,Austria) with excitation at 485 nm and emission at 535 nm. Thepercentage (%) specific lysis was calculated as: (test calceinrelease−spontaneous calcein release)×100/(maximal calceinrelease−spontaneous calcein release). Maximal lysis was achieved byadding 1% TRITON X-100.

The results are shown on FIG. 10.

Legend of FIG. 10: Calcein-AM loaded target cells JOK1.5.3 wereincubated in presence or absence of 1 or 5 μg/ml bi-specific MUT 4, CR3or rituximab (RTX) antibodies and in presence of CIK cells at a 10:1effector:target ratio. After 4 hours, supernatants were collected andreleased calcein measured. The data show the measured percentage lysis(Y-axis) as means and standard deviations of 2 independent experiments.−: control without antibody.

The results show that in vitro, percentage killing (lysis) of HLADRpositive JOK15.3 is increased by 60-70% by addition of 1-5 μg/ml MUT 4antibody and by 40-50% by addition of CR3 antibody, in presence of CIKcells at a 10:1 effector:target ratio. Rituximab in contrast has nosignificant effect. This demonstrates that the intercellular bridgeformed by the bi-specific MUT 4 antibody dramatically enhances killingof the HLADR⁺ targets by CIK cells, similar or higher to that obtainedwith CR3.

The invention claimed is:
 1. An isolated multispecific antigens-binding fragment comprising at least two Fab fragments with different CH1 and CL domains, wherein said Fab fragments comprise: a. a wild-type Fab fragment consisting of wild-type human CH1 and wild-type human CL domains of an immunoglobulin, and the VH and VL domains of an antibody recognizing an epitope of interest; and b. a mutated Fab fragment consisting of: i. the VH and VL domains of an antibody recognizing an epitope of interest; ii. a CH1 domain of a human immunoglobulin comprising substitution of the threonine residue at position 192 of said CH1 domain with a glutamic acid residue ; and iii. a CL-kappa domain of a human immunoglobulin comprising substitution of the asparagine residue at position 137 of said CL domain with a lysine residue and substitution of the serine residue at position 114 of said CL domain with an alanine residue, wherein the sequence position numbers used for the CH1 and CL domains refer to Kabat numbering and each Fab fragment recognizes a different epitope of interest, and said Fab fragments are tandemly arranged in any order, the C-terminal end of the CH1 domain of the first Fab fragment being linked to the N-terminal end of the VH domain of the following Fab fragment through a polypeptide linker.
 2. The isolated multispecific antigens-binding fragment of claim 1, wherein the polypeptide linker has a length of at least 20 amino acids.
 3. The isolated multispecific antigens-binding fragment of claim 1, wherein the polypeptide linker comprises all or part of the sequence of the hinge region of one or more immunoglobulin(s) selected from the group consisting of: IgA, IgG, and IgD.
 4. The isolated multispecific antigens-binding fragment of claim 1, wherein the polypeptide linker has the sequence of SEQ ID NO:
 9. 5. An isolated multispecific antibody having two identical antigens-binding arms, each consisting of an antigens-binding fragment of claim
 1. 6. The isolated multispecific antibody of claim 5, which has an immunoglobulin-like structure, comprising: a. two identical antigens-binding arms each consisting of an antigens-binding fragment of any of claims 1 to 4; b. the dimerized CH2 domains and CH3 domains of an immunoglobulin; and c. the hinge region of an IgA, IgG, or IgD, linking the C-terminal ends of the CH1 domains of the antigens-binding arms to the N-terminal ends of the CH2 domains.
 7. A therapeutic composition comprising: a. a multispecific antigens-binding fragment of claim 1 and a pharmaceutically acceptable vehicle, or b. a multispecific antibody of claim 5, and a pharmaceutically acceptable vehicle. 